NADPH Selective Depletion Nanomedicine‐Mediated Radio‐Immunometabolism Regulation for Strengthening Anti‐PDL1 Therapy against TNBC

Abstract Anti‐PD(L)1 immunotherapy recently arises as an effective treatment against triple‐negative breast cancer (TNBC) but is only applicable to a small portion of TNBC patients due to the low PD‐L1 expression and the immunosuppressive tumor microenvironment (TME). To address these challenges, a multifunctional “drug‐like” copolymer that possesses the auto‐changeable upper critical solution temperature and the capacity of scavenging reduced nicotinamide adenine dinucleotide phosphate (NADPH) inside tumor cells is synthesized and employed to develop a hypoxia‐targeted and BMS202 (small molecule antagonist of PD‐1/PD‐L1 interactions)‐loaded nanomedicine (BMS202@HZP NPs), combining the anti‐PD‐L1 therapy and the low‐dose radiotherapy (LDRT) against TNBC. In addition to the controlled release of BMS202 in the hypoxic TNBC, BMS202@HZP NPs benefit the LDRT by upregulating the pentose phosphate pathway (PPP, the primary cellular source for NADPH) of TME whereas scavenging the NADPH inside tumor cells. As a result, the BMS202@HZP NPs‐mediated LDRT upregulate the PD‐L1 expression of tumor to promote anti‐PD‐L1 therapy response while reprogramming the immunometabolism of TME to alleviate its immunosuppression. This innovative nanomedicine‐mediated radio‐immunometabolism regulation provides a promising strategy to reinforce the anti‐PD‐L1 therapy against TNBC.

The protocol for animal experiments was approved by the Animal Experimentation Ethics Committee of School of Life Science and Technology in Xi'an Jiaotong University. Female Balb/c mice (5-6 weeks) were purchased and maintained in the Center for Experimental Animals at Xi'an Jiaotong University Health Science Center. Before X-ray radiation treatment, each mouse was anesthetized and shielded by custom lead blocks, such that only the irradiated tumor was exposed. Radiation was performed using a digital accelerator (Elekta Precise) operated at 6 MV, and a dose rate of 600 MU min -1 .
The tumor volume was measured via a serial caliper and estimated using Equation 1: 2 (1) The tumor inhibitory efficiency was calculated by Equation 2: where V Sample and V 0.9% NaCl represented the tumor volume of the mice treated with different drug formulations and 0.9% NaCl, respectively.

Turbidity measurements of PAAN copolymer
To demonstrate the changeable UCST of PAAN copolymer under hypoxia, the PAAN and PAA copolymer were dispersed in aqueous solution containing NADPH (1mM) and NTR (10 μg mL -1 ) and incubated at 37 o C under argon protection for 0, 12 and 24 h, and their turbidities were recorded using a UV-vis spectrophotometer (Purkinje T6-1610F, China) at a wavelength of 650 nm and a heating rate of 0.5 o C min -1 . The UCST of copolymer was defined as the temperature where the transmittance reached half of the maximum value during the whole heating process. In addition, the UV absorbance of PAAN solutions before and after incubation in different solutions was also monitored using a UV-vis spectrophotometer.

Electron affinity measurements of PAAN copolymer
Cyclic voltammetry measurement was employed to evaluate electron affinity of NIEAAm or PAAN copolymer with an equal concentration of nitroimidazole ([NI] = 0.002 M). Meanwhile, n-Bu 4 NPF 6 (0.2 M) was selected as the supporting electrolyte. Besides, the Ag/AgCl and platinum electrodes were used as the reference and working electrodes, respectively. The scanning rate was 100 mV s -1 .

Preparation and Characterization of HZP NPs
The blank HZP NPs with dual targeting ligands (HA and Z) were fabricated using a modified emulsification technique. Briefly, PAAN copolymer dissolved in DMSO (250 μL, 20 mg mL -1 ) was injected into 1 mL of HA-HDA-Z aqueous solution (1 mg mL -1 ) under probe sonication at 14 W for 1 min on ice (Sonics & Materials, VCX500). Then, the resultant emulsion was transferred into a dialysis bag (MWCO 3500 Da) and dialyzed against deionized water overnight. The blank HP NPs with single targeting ligand (HA) was prepared using a similar procedure except that HA-HDA-Z was replaced with HA-HDA. Meanwhile, to evaluate their drug-loading properties, the targeting capacities and in vivo biodistribution of HZP NPs, the drugs or the fluorescent dyes-loaded NPs were also prepared using the same protocol for blank NPs fabrication, except that the drugs (0.1 mg BMS202) or the fluorescent dyes (0.025 mg coumarin 6 (C6) or 0.15 mg DiR) were co-added to the DMSO solution containing PAAN or PAA copolymer during the HZP or HP NPs preparation, respectively.
Afterward, the size and zeta potential of blank HZP NPs as well as their stability in simulated physiological condition were evaluated using a dynamic light scattering (DLS) analyzer (ZETA-SIZER, NanoZS90, Malvern, Ltd., UK). Meanwhile, the morphology of HZP NPs was observed using a transmission electron microscope (FEI Talos F200C, US).

Measurement of HZP NPs' targeting capacity
4T1 cells at a density of 1.5 × 10 5 cells well -1 were seeded in 24-well plates overnight, and

Entrapment efficiency of BMS202 encapsulated in HZP NPs
The content of BMS202 encapsulated in HZP NPs was determined by high performance liquid chromatography (HPLC) using a C18 column (25 mm × 4.6 mm, 5 μm) on Agilent 1100 system. The column was eluted using a mixture of different gradient ratios of acetonitrile and water at 30 o C with a flow rate of 1.0 mL min -1 . The content of BMS202 was monitored with a UV detector according to their absorbance at 230 nm. The entrapment efficiency of BMS202 loading in NPs was calculated using Equation 3: Entrapment efficiency (%) = drug amount in NPs / amount of drug used ×100% (3)

In vitro drug release profiles
To investigate in vitro drug release profiles, BMS202@HZP NPs were added into dialysis bags (MWCO:14 kDa) and then immersed into PBS (pH 7.4, 0.01 M) at 37 ℃. In addition, NADPH (1 mM) and NTR (10 μg mL -1 ) was added in external solution under argon protection to mimic hypoxic condition. At predetermined time intervals, the external solution was removed and the dialysate was replenished by fresh solution with the same volume. The amount of released BMS202 was determined using HPLC as described above.

Blocking PD-L1 receptor efficacy
Flow cytometry measurement was utilized to quantitatively evaluate the affinity of BMS202@HZP NPs with the PD-L1 protein. Briefly, 4T1 cells at a density of 3 × 10 5 cells well -1 were seeded in 6-well plates for 24 h. Then, the cells were treated with free BMS202 and BMS202@HZP NPs ([NI] = 238 μM, [BMS202] = 2.57 μg mL -1 ), which were incubated for another 24 h under hypoxic conditions. Subsequently, the cells were washed with PBS, then incubated with PE-anti-PD-L1 followed by flow cytometry detection.

Selective NADPH depletion
To evaluate the nanomedicine's capacity of selective scavenging NADPH inside tumor cells, 4T1 cells and RAW264.7 cells at a density of 6 × 10 5 cells well -1 were seeded in 6-well plates μg mL -1 ) for 6 h under hypoxic and normoxic conditions, followed by 0 or 2 Gy of X-ray radiation. After another 24 h of incubation, 4T1 cells were collected to evaluate the CRT expression using western blot. The intensities of bands were quantified by ImageJ.

Colony formation assay
For colony formation assay, 4T1 cells at a density of 800 cells well -1 were seeded in 6-well plates for 24 h. Then, the cells were treated with free BMS202, blank HZP NPs and BMS202@HZP NPs ([NI] = 238 μM, [BMS202] = 2.57 μg mL -1 ) for 6 h under hypoxic and normoxic conditions, which were then exposed to X-ray radiation at a dose of 2 Gy.
Additionally, the experiment was performed for 7 days and the media were changed every other day. At the end of experiment, the cell clones were immobilized with methanol, stained with crystal violet and photographed. Finally, the stained cells were decolorized with 30% acetic acid, and the absorbance of decolonization solution at 540 nm wavelength was measured using a microplate reader (Tecan M200) to quantitatively estimate the cell clone rate.

In vitro immune regulation
To Meanwhile, the supernatant in the lower chamber was also collected for TNF-α and IFN-γ determination using ELISA assay.

In vivo biodistribution studies
For HZP NPs' biodistribution evaluation, subcutaneous 4T1 tumor were cultured on the back of female mice. When the tumor size reached to 400∼ 500 mm 3 , the 4T1 tumors were treated with or without 2Gy X-ray radiation followed by intravenous injection of free DiR and DiRloaded HZP and HP NPs at the dye dose of 0.35 mg kg -1 mouse body weight. At predetermined time intervals (2,4,6,8,12, and 24 h), the fluorescent images of these mice were captured using in vivo imaging system (MAG Biosystems Lumazone, USA). At the end of experiment, the mice were sacrificed and the major tissues (heart, liver, spleen, lung, kidney and tumor) were harvested for ex vivo imaging.
Additionally, the tumor-bearing mice pre-treated with or without 2 Gy of X-ray radiation were injected (i.v.) with 0.9% NaCl, BMS202@HP NPs and BMS202@HZP NPs at a dose of 1 mg BMS202 per kg body weight of mouse, respectively. At 24 h post-injection, the mice were immediately injected with Evans blue solution. After another 24 h, all mice were subjected to PBS perfusion, and tumor tissues were harvested and photographed. Finally, all tumor tissues were dried and weighed, and the Evans blue was then extracted from tumor using DMF, which was quantified by determining the absorbance at 620 nm.
Different drug formulations were injected (i.v.) into Balb/c mice on day 1, 4, and 7. At 24 h post-injection, the tumors were exposed to X-ray at a dose of 2 Gy. The tumor size, body weight, and survival rate were monitored throughout the whole experiment period. Besides, on day 10, a part of mice in each group was sacrificed, and the tumors and plasma were collected to evaluate the immune responses.
For RNA sequencing analysis, total RNA from tumor tissues was extracted. Libraries were subsequently constructed using Standard Illumina Novaseq. Transcriptome sequencing and analysis were performed by Haorui Genomics Co, Ltd (Xi'an, China). Besides, the tumors were collected to evaluate the mechanisms of immune regulation. Briefly, the metabolites of tumor tissues were analyzed by gas chromatography-mass spectrometry (GC-MS). Briefly, the extraction buffer (methanol/water/chloroform = 2.5:1:1) was added to tumor tissues at a ratio of 1:10 (w/v). Subsequently, the extracts were subjected to oximation and derivatization.
Finally, the metabolites in tumor tissues were measured by Agilent 7890A gas chromatograph and 5975C mass spectrometer (Agilent Technologies, Wilmington, DE). In addition, the enzymatic activities of G6PD and 6PGD and the level of NADPH and NADP + in tumor were also measured according to the manufacturer's protocols of assay kits.

Abscopal effect and anti-lung metastasis efficacy
The 4T1 bilateral tumor model was established to evaluate abscopal effect and anti-lung metastasis efficacy of BMS202@HZP NPs-mediated LDRT. Briefly, 1 × 10 6 4T1 cells were injected (s.c.) in the right flank of Balb/c mice as primary tumor. 7 days later, 1 × 10 5 4T1 cells were injected in the left flank of Balb/c mice as distant tumor. When the primary tumors reached ~150 mm 3 , all mice were randomly divided into five groups with the same treatment as mentioned above, and the primary tumor was treated with 2 Gy of X-ray radiation. The volumes of primary and distant tumors were monitored throughout the whole experiment period. At the end of experiment, the primary and distant tumor, spleen and plasma were collected for H&E, IHC analysis (CD8, granzyme B and CD20) and ELISA assay (IFN-γ and TNF-α), respectively. Besides, the lungs were also harvested and stained by Bouin's solution (picric acid/formaldehyde/acetic acid = 15:5:1) and H&E to further evaluate the anti-lung metastasis efficacy.

Biosafety studies
To study the biosafety of NPs, the hemolysis tendency of blank HZP and HP NPs was evaluated. Briefly, the erythrocytes at the density of 1×10 7 cells mL -1 were incubated with blank HP NPs and HZP NPs at concentrations ranging from 50 μg mL -1 to 500 μg mL -1 at 37 ℃ for 12 h. PBS (pH 7.4, 0.01 M) and Triton X-100 (1%, v/v) solution were also selected as negative and positive control, respectively. The absorbance of hemoglobin at the wavelength of 410 nm in the supernatant was measured using the microplate reader (Tecan M200). Hemolytic activity (%) was calculated using Equation 5: where A Sample , A PBS , A Triton represent the absorbance intensity of hemoglobin in blank HZP and HP NPs, PBS, and Triton X-100, respectively.
Furthermore, the biosafety of free BMS202 and BMS202@HZP NPs were also estimated in vivo. In brief, the major organs (heart, liver, spleen, lung and kidney) from mice treated with free BMS202 and BMS202@HZP NPs were harvested and stained with H&E after 21 days of treatment. Additionally, the blood was also collected for the routine blood examination and the plasma was obtained for biochemistry analysis, including aspartate transaminase (AST), creatinine (CRE), alanine aminotransferase (ALT), and urea nitrogen (BUN). Meanwhile, the metabolomics of serum was also analyzed by GC-MS using the same treatment as mentioned above, except that the extraction buffer was a mixture of isopropanol, acetonitrile and water in a ratio of 3:3:2 (v/v/v).

Statistical analysis
All of the data are reported as the mean ± standard deviations (SD) from at least three repeated experiments. Statistical analysis were performed using a two-sided Student's t-test unless otherwise indicated (Graphpad Software). *p < 0.05 and **p < 0.01 were considered to be statistically significant and extremely significant, respectively.      tumor after the treatments of 0.9% NaCl, BMS202@HP NPs and BMS202@HZP NPs with or without 2 Gy of X-ray radiation. (mean ± SD, n = 3, *p < 0.05, **p < 0.01) Figure S11. IHC staining for PD-L1 of tumor tissue in Balb/c mice bearing 4T1 tumors following the treatments of 0.9% NaCl with or without 2 Gy of X-ray radiation and BMS202@HZP NPs with 2 Gy of X-ray radiation.

Figure S12. H&E images and IHC staining of CD8+ T cell and Gran B of tumor tissue in
Balb/c mice bearing 4T1 tumors following the treatments of BMS202@HP NPs, 0.9% NaCl and BMS202@HZP NPs with or without 2 Gy of X-ray radiation. Figure S13. Tumor inhibitory efficiency in Balb/c mice bearing 4T1 tumors following the treatments of 0.9% NaCl with 2 Gy of X-ray radiation, BMS202@HP NPs and BMS202@HZP NPs with or without 2 Gy of X-ray radiation. (means ± SD, n = 5, *p < 0.05, **p < 0.01) Figure S14. H&E images of distant tumors in Balb/c mice bearing 4T1 bilateral tumors following the treatments of BMS202@HP NPs, 0.9% NaCl and BMS202@HZP NPs with or without 2 Gy of X-ray radiation.  Balb/c mice after systematic injection of 0.9% NaCl, free BMS202 and BMS202@HZP NPs.